This proposal has three parts all of which focus on the transduction of either normal or pathological signals to the microtubules in Alzheimer's disease. In contrast to the identified signaling pathways that affect actin organization, very little is known about how signals are conveyed to microtubules. The hypothesis of part I is that the projection domains of microtubule-associated proteins (MAPs) serve as anchors for specific kinases and phosphatases where they modulate local alterations in microtubule organization following upstream activation. Kinases and phosphatases that interact with the projection domain of MAP2 and tau will be identified and the interaction characterized. In preliminary work using coimmunoprecipitation techniques we found a physical association between the mitogen-activated protein kinase (MAPK) and both MAP2 and tau. Protein phosphatase 2A and cdk5 were bound only to MAP2 and not to tau. To test the effects of phosphorylation on tau function we will utilize expression in Sf9 cells because in these cells tau induces a highly ordered and specific cytoskeletal organization. Sf9 cells expressing recombinant tau which has been mutated at in vivo phosphorylation sites will be morphometrically analyzed for process formation, microtubule stability, and microtubule spacing. To connect tau modifications to a specific kinase we have cloned an inhibitory MAPK sequence into (a) baculoviral cassette that expresses tau from the polyhedrin promoter and the MAPK inhibitory sequence from the plO promoter; and into (b) a eukaryotic expression vector with an epitope tag for expression in PC12 cells. The effects of this construct on cell morphology, on tau phosphorylation, and on other putative MAPK substrates will be determined. Part II addresses a signaling cascade we have identified in which APP2 activates MAPK and results in the increased phosphorylation of tau. A cysteine-rich region at the amino terminus of APP is responsible for this activation. Our preliminary data suggests that in contrast to NGF, APP2 activation requires cell attachment. The presence of cysteines in the active site and the requirement for cell attachment have led us to hypothesize a mechanism of activation similar to basic fibroblast growth factor which requires heparin for high affinity binding to the receptor. An alternative hypothesis is addressed in experiments which will assess whether the association of APP with caveolae represents a signal transduction pathway. Part III focuses on pathological signals affecting the organization of the cytoskeleton. Particularly useful will be one month old hippocampal cultures in which we have shown mature tau splicing. Following treatments with combinations of A-beta, APP2, and apoE we will study the tau phosphorylation state and look for PHF-like structures. Because the possibility of apoE transport to the cytosol has been raised as part of the pathogenetic schema, we will determine whether apoE reaches the cytosol by conjugating apoE to saporin and adding the conjugate to cultures. Saporin is toxic to cells only when it reaches the cytosol.